Dna sequence that increases odorant receptor representation in the olfactory system

ABSTRACT

A genetically modified vertebrate is provided that has an enhanced sense due to an over representation of a predetermined odorant receptor. The vertebrate is genetically modified by introduction of DNA that comprises at least four sequential repeats of a sequence whose primary structure is at least 90% homologous with ACATAACTTTTTAATGAGTCT (SEQ ID NO: 1). The DNA causes a nearby odorant receptor coding sequence to be over represented in a singular gene choice fashion relative to a corresponding vertebrate that lacks the DNA.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and is a non-provisional of U.S.Patent Application 62/200,312 (filed Aug. 3, 2015) and 62/312,068 (filedMar. 23, 2016) the entirety of which are incorporated herein byreference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numberGM088114-01A1 awarded by the National Institute of Health and grantnumber MD007599 awarded by the National Institute on Minority Health andHealth Disparities. The government has certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document entitled “Sequence.txt” (5 kb createdon Aug. 3, 2016) which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

The subject matter disclosed herein relates to generating geneticallymodified organisms and, in particular, to genetically modified organismswith an increased representation of odorant receptors, whichconcomitantly enhances the sense of smell. Currently, animals such asdogs, bees and rats are deployed to help humans with scent detectiontasks, but their use is expensive since they require long-term training.Having a genetically manipulated organism with an increasedrepresentation of specific odorant receptors will considerably shortenthe training process and advance animal-based scent detection tasks.Having a genetically manipulated organism with an increasedrepresentation of specific odorant receptors increases researcher'sability to analyze odors that activate that particular odorantreceptors.

Volatile chemicals are detected by several million olfactory sensoryneurons (OSNs) arrayed in a sensory epithelium located inside the nasalcavity. The main olfactory epithelium (MOE) expresses odorant receptors(ORs) through a poorly understood singular gene choice mechanism wherebyonly one allele of any OR gene is selected for specific expression in agiven neuron. Typically, ˜0.1% of the total number of OSNs in the MOEexpresses the same OR and their axons coalesce into homotypic glomeruliin the olfactory bulb, the first relay station for synaptic activity inthe brain. Thus, all ORs being expressed in equal and low representationmakes the olfactory neuronal sheet a broad, non-specific detector ofodorants.

Several million olfactory sensory neurons (OSNs) in the nose are used toidentify volatile chemicals (odors). Rodents and dogs carry about 1000individual odorant receptor (OR) genes, whose proteins can bind tospecific odors; roughly 10,000 neurons are associated with an individualOR gene. Thus, each OR is present in an equal and low representation(0.1%), which makes the olfactory neuronal sheet a broad, non-specificdetector of odorants.

There has been limited success in odor profiling ORs expressed inheterologous cells in vitro. Part of this limitation is due to theinability of OR proteins to traffic to the plasma membrane. In addition,given the biological properties of the olfactory system many in vitrocharacterized OR alleles may not be functional in an in vivo setting.The major drawback, though, has been the ability to rapidly contrast howodors presented in liquid phase (in vitro) correspond to odors presentedto the OR in vapor phase within their mucosal environment (in vivo).Even ex vivo patching of dendritic knobs from transgenic andgene-targeted mice suffers from an absence of vapor phase delivery ofodors. In addition, the study of both OR gene choice and OR coding invivo is hampered by the low representation of a given OR, i.e. only˜0.1% on average of the total neuronal population in a wild type mouse.A rapid in vivo approach to odor profile any OR would be a breakthroughand permit researchers to correlate in vitro, ex vivo, and in vivoresponses. Unfortunately, no suitable in vivo approach is known.

The discussion above is merely provided for general backgroundinformation and is not intended to be used as an aid in determining thescope of the claimed subject matter.

BRIEF DESCRIPTION OF THE INVENTION

A genetically modified vertebrate is provided that has an enhanced sensedue to an overexpression of a predetermined odorant receptor. Thevertebrate is genetically modified by introduction of DNA that comprisesat least four sequential repeats of a sequence whose primary structureis at least 90% homologous with ACATAACTTTTTAATGAGTCT (SEQ ID NO: 1).The DNA causes a nearby odorant receptor coding sequence to be overrepresented in a singular gene choice fashion relative to acorresponding vertebrate that lacks the DNA.

In a first embodiment, a method of producing a genetically modifiednon-human vertebrate is provided. The method comprises inserting DNAinto a genome of a non-human vertebrate using a vector. The vectorcomprises a M71 odorant receptor (OR) transgene backbone; at least threesequential repeats of a DNA sequence with two complete ds B-DNA turns,the DNA sequence being at least 90% homologous with 5′ACATAACTTTTTAATGAGTCT 3′ (SEQ ID NO: 1); a transcription start site(TSS) disposed downstream of the at least three sequential repeats; anodorant receptor coding sequence disposed downstream of thetranscription start site, the odorant receptor coding sequence codingfor a predetermined odorant receptor; wherein the non-human vertebrate,after the step of inserting, develops a main olfactory epithelium (MOE)comprising multiple olfactory sensory neurons (OSNs), wherein between10% and 95% of the OSNs express the predetermined odorant receptor.

In a second embodiment, a vector for genetically modifying a vertebrateis provided. The vector comprises a M71 odorant receptor (OR) transgenebackbone; at least three sequential repeats of a DNA sequence that is atleast 90% homologous with 5′ ACATAACTTTTTAATGAGTCT 3′ (SEQ ID NO: 1); atranscription start site (TSS) disposed downstream of the at least threesequential repeats; an odorant receptor coding sequence disposeddownstream of the transcription start site, the odorant receptor codingsequence coding for a predetermined odorant receptor; wherein thevertebrate develops an olfactory epithelium that expresses between 10%and 70% of the predetermined odorant receptor.

In a third embodiment, a genetically modified non-human vertebrate isprovided. The vertebrate comprises germ cells and somatic cells thatcontain an insertion of DNA within a 10 kb proximity of a transcriptionstart site (TSS) of an odorant receptor transgene, such that theinsertion causes the vertebrate to over represent a predeterminedodorant receptor corresponding to the odorant receptor transgenerelative to a corresponding vertebrate that lacks the insertion, whereinthe DNA comprises at least three sequential repeats of a DNA sequencewith two complete ds B-DNA turns whose primary structure is at least 90%homologous with ACATAACTTTTTAATGAGTCT (SEQ ID NO: 1).

In a fourth embodiment, a method of high-throughput screening for anodorant ligand is provided. The method comprising steps of: exposing acandidate odorant ligand to an olfactory neuron or cilia of a biosensor,the biosensor comprising a plurality of cells with neurons in anolfactory epithelium wherein greater than 1% of the neurons in theolfactory epithelium are expressing an odorant receptor (OR) through asingular gene choice enhancer nucleotide sequence with at least threerepeats of a DNA sequence that is at least 90% homologous with 5′ACATAACTTTTTAATGAGTCT 3′, the at least three repeats being disposedwithin 10 kb from a predetermined odorant receptor; and monitoring theolfactory neuron or the cilia for a change in expression of GDP, cAMPand/or ATP, wherein an increase corresponds to successful binding of thecandidate odorant ligand to the predetermined odorant receptor.

This brief description of the invention is intended only to provide abrief overview of subject matter disclosed herein according to one ormore illustrative embodiments, and does not serve as a guide tointerpreting the claims or to define or limit the scope of theinvention, which is defined only by the appended claims. This briefdescription is provided to introduce an illustrative selection ofconcepts in a simplified form that are further described below in thedetailed description. This brief description is not intended to identifykey features or essential features of the claimed subject matter, nor isit intended to be used as an aid in determining the scope of the claimedsubject matter. The claimed subject matter is not limited toimplementations that solve any or all disadvantages noted in thebackground.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the features of the invention can beunderstood, a detailed description of the invention may be had byreference to certain embodiments, some of which are illustrated in theaccompanying drawings. It is to be noted, however, that the drawingsillustrate only certain embodiments of this invention and are thereforenot to be considered limiting of its scope, for the scope of theinvention encompasses other equally effective embodiments. The drawingsare not necessarily to scale, emphasis generally being placed uponillustrating the features of certain embodiments of the invention. Inthe drawings, like numerals are used to indicate like parts throughoutthe various views. Thus, for further understanding of the invention,reference can be made to the following detailed description, read inconnection with the drawings in which:

FIG. 1 depicts a design for a transgene using the disclosed DNAsequence;

FIGS. 2A to 2C depict odor response profiles of in vivo GlomerularImaging of M71 biosensors with 4.M71Cα lines, wherein FIG. 2A showsacetophenone (ACP), FIG. 2B shows 4-methyl acetophenone (4MACP) and FIG.2C shows 2,4-dimethyl acetophenone (24dMACP); and

FIGS. 3A, 3B and 3C are graphs depicting the results of behavioravoidance assays on 4.M71Cα homo lines with 2,4-dimethyl acetophenone(24dMACP) (FIG. 3A); 5.M71Cα lines with 2,4-dimethyl acetophenonen(24dMACP) (FIG. 3B) and 5.OR1A1Cβ lines with (−)-Carvone (FIG. 3C).

DETAILED DESCRIPTION OF THE INVENTION

This disclosure pertains to a method of controlling the expression ofspecific odorant receptors in vivo in mice by multimerizing a specific21 bp sequence encompassing the homeodomain binding site sequence,TAATGA, known to be a determinant in OR gene choice and adding it to theM71 odorant receptor (OR) transgene backbone (see Rothman et al. ThePromoter of the mouse odorant receptor gene M71; Mol. Cell. Neurosci.28, 535-546, 2005). Of note, the 21 bp sequence was chosen to reflecttwo complete turns of double stranded B-DNA (10.5 bp per turn onaverage). The method reproducibly and dramatically increases the totalnumber of olfactory sensory neurons (OSNs) expressing specific mouse orhuman OR coding sequences in different transgenic animals. Thetransgenic vector was designed in such way that any odorant receptor orG-protein-coupled receptors (GPCR) (human, mouse or from any species),can be shuttled into the transgenic backbone. Importantly, proof ofconcept studies show that transgenic mice with over-represented ORs showan increased sensitivity to cognate odors in odor detection behavioraltasks. As such, a versatile platform has been created that providesbiosensors by genetically manipulating gene expression in the mouse'snose with great translational potential. The platform permits researchesto study gene choice, axon identity and odor coding simultaneously invivo, three processes that cannot be studied independently from eachother.

To up-regulate the neuronal population expressing a given OR a methodthat generates transgenic mice in which 10-70% of the mouse olfactoryepithelium expresses the same OR that has been labeled with a redfluorescent protein. An expression system was developed using the M71 (amouse OR) transgene backbone containing a strong tunable enhancer placedupstream of any OR coding region. The tunable enhancer consists ofrepeats of a specific 21-mer sequence (hereafter referred to as ×21): 5′ACATAACTTTTTAATGAGTCT 3′ (SEQ ID NO: 1). This sequence contains theTAATGA sequence, which is correlated with singular gene choice events.Usually one OSN expresses only one OR. Once a given OSN has ‘chosen’ itsOR the OSN does not choose another OR. Using the disclosed method, agiven OSN system is forced to choose a predetermined OR of interest withgreater probability.

Biosensors expressing the mouse M71 OR and the human OR1A1 OR have beenvalidated, both on a molecular level and a functional level. Without useof the disclosed method about 0.1% of the total number of olfactorysensory neurons in the main olfactory epithelium express the sameodorant receptor (OR) in a singular fashion and their axons coalesceinto homotypic glomeruli in the olfactory bulb. In proof-of-principlestudies the expression of a mouse OR (M71) has been increased by 45-fold(about 1-2%) to 100,000-200,000 OSNs and a human OR (OR1A1) has beenincreased by 500-fold (about 13%) to 1.3 million OSNs, while conservingthe biological properties of the olfactory system. The transgenicanimals have enhanced brain activity to known odors recognized by thosespecific ORs, as shown by live animal in vivo imaging. Importantly,improved odor detection seems to translate to the higher corticalregions, since the transgenic animals show increased sensitivity totheir cognate odors in behavioral odor avoidance assays.

Downstream of the OR coding sequence an internal ribosomal entry site(IRES) is inserted followed by the coding sequence for a red fluorescentprotein (tauCherry). This allows for bicistronic translation and, assuch, co-expression of the OR of interest with tau-Cherry, enablingvisualization of the over represented ORs in vivo using fluorescencemicroscopy. In one embodiment, the fluorescent protein is omitted whenvisualization is not desired.

Detailed Results

OR genes form the largest multigene family in mammals with about 1200members in the mouse and about 350 members in humans. The main olfactoryepithelium (MOE) expresses ORs through a poorly understood singular genechoice mechanism whereby only one allele of any odorant receptor gene isselected for specific expression in a given neuron. Axons from OSNs thatexpress identical ORs coalesce into two out of the ˜4800 glomeruli inthe olfactory bulb (OB). The odorant receptor coding sequence (OR CDS)plays a role in the maintenance of gene choice. If the OR is not capablefor this maintenance, then a second OR allele is tested forfunctionality. Hence, deletion of an OR CDS precludes the convergence ofaxons into a specific glomerulus and results in OSNs choosing to expressother OR genes projecting to a variety of glomeruli in the OB. Inaddition, OR proteins are necessary for promoting axon guidance, axonidentity and stabilizing neurons that have chosen to express those ORs.Finally, the OR protein needs to be targeted to the olfactory ciliawhere it will function in odor signal transduction. As used in thisspecification, the term odorant receptor coding sequence (OR CDS) refersto a sequence of DNA that promotes the growth of an individual odorantreceptor.

In the case of a M71 OR minigene, a 7.5 kb DNA fragment accuratelyrecapitulates the functionality of the gene-targeted M71 locus andimparts an expression pattern paralleling that of endogenous genes. Twohighly conserved sequences were identified: (1) a single candidate O/Ebinding site and (2) a two candidate-Lhx2 homeodomain (HD) binding siteswithin this 161 bp region. An HD sequence is believed to be highlydesirable for regulating the probability for any OR gene to beexpressed.

Further analysis of sequences known to strongly influence OR gene choicesuch as the mouse H (the Homology region that activates the MOR28cluster, and P (a sequence with high homology to the P3 minimalpromoter, elements have revealed a set of three HD binding sites inclose proximity to each other, and an associated O/E site, with one ofthe HD sites sharing the same 13mer AACTTTTTAATGA (SEQ ID NO: 2) betweenthem. When a 19mer (see SEQ ID NO: 10, FIG. 1) containing this 13mersequence from P element was multimerized nine times (9×19) and placedupstream of the MOR23 transgene backbone, inconsistent increases in cellnumbers were observed.

The disclosed enhancer was designed to accommodate two full turns of thedouble helix using a multimerized 21 bp HD encompassing 13mer sequencefrom the “H element” resulting in a radical and reliable increase inexpression of any odorant receptor protein in the context of the M71transgene backbone. The disclosed method permits researchers, for thefirst time, to study OR gene choice, axon identity and odor codingsimultaneously in its intact in vivo environment.

The Transgene

The effect of a multimerized 21-mer of the H element (DNA turns every10.5 bp thereby allowing for maximum cooperativity) was tested on theprobability of choice for any OR CDS expressed from the M71 transgene.Using the M71 transgene backbone including 485 bp of the M71 promoterupstream of the transcription start site (TSS) (FIG. 1), a modularversion of the transgenic vector was created such that any number of21mer repeats can be shuttled into the NheI site at position −485. Themodular version of the transgenic vector is a modified form of the M717.5 kb minigene disclosed by Rothman (The Promoter of the mouse odorantreceptor gene M71; Mol. Cell. Neurosci. 28, 535-546, 2005) wherein theM71 OR CDS has been replaced with an OR CDS of choice. The transgenebackbone refers to the disclosed minigene without the M71 OR CDS.

Any OR CDS of interest can be cloned into the AscI site followed by, ifdesired, a label such as an IRES-tauCherry cassette. The internalribosomal entry site (IRES) allows for bicistronic translation andsimultaneous expression of tauCherry enabling the visualization of theolfactory neuronal morphology.

FIG. 1 specifically depicts a design of a transgene. The transgenicvector was created using the M71 OR genomic backbone. Any number of H_21mer (×21) repeats can be shuttled in the NheI site upstream of the TSSand any OR CDS can be cloned into the AscI site. An IRES− tauCherrycassette (Pad) follows the OR CDS. The internal ribosomal entry sequence(IRES) allows for bicistronic translation and simultaneous expression ofmCherry enabling the visualization of the olfactory morphology.Transgenic animals were generated by pronuclear injection of PmeI(black) digested DNA. In one embodiment the repeats are disposed within10 kb of a predetermined OR CDS. In another embodiment, the repeats aredisposed at least 500 bp from a predetermined OR CDS but less than 10 kbfrom the predetermined OR CDS. In one embodiment the repeats aredisposed upstream of the OR CDS. In another embodiment, the repeats aredisposed downstream of the OR CDS.

Effect of 21mer Multimerization

An initial analysis of various M71 transgenic lines containing 0, 1, 2,3 or 4 multimers of the 21mer sequence was performed (Table 1).

Founds with OSN expression¹/ Germline Established with OSN Robust OSNgermline mouse OR Enhancer expression Expression² line Mouse OdorantReceptors M71 0x21 0.14³/1:2 NO NA M71 D205E 1x21 NA/0:1 NO NA M71-D205L2x21 NA/2:3 NO NA M71-D205N 3x21 NA/1:1 NO NA M71 4x21 NA/3:3 YES4·M71Cα 4·M71Cβ M71 5x21   6:6/1:1 YES 5·M71Cα MOR122-2 5x21 NA/5:5 YES5·MOR122-2Cα 5·MOR122-2Cβ 5·MOR122-2Cδ Human Odorant Receptors OR1A15x21 NA/2:2 Yes 5·OR1A1Cβ 5·OR1A1Cδ ¹Positive Founders at 20 months ofage ²Presence of stable glomeruli that are bigger than 30 μm(M71-IRES-tauGFP glomeruli) ³Small glomeruli are unstable in olderanimals.

This data shows that less than 50% germline animals containing fewerthan four 21 multimers show expression of the cloned OR and none of themare robust expressers. On the contrary, 4×21 M71 lines generated robustexpression in 100% of germline animals (3/3). Two 4×21-M71 lines(4.M71Cα and β) were bred for further analysis. In addition, the copynumber of the transgenes insertions were assessed to determineinfluences the rate of OR expression by quantitative real-time PCR(qPCR) and found no correlation between the expression level and copynumber. Hemizygous 4.M71Cα contain about 15 copies whereas hemizygous4.M71Cβ showed about 24 copies. Interestingly, nine 0.M71C founderscontained between 5-32 insertions of the transgene, but do not show anyrobust OR expression. Without wishing to be bound to any particulartheory, it is believe the 4×21 enhancer is able to modulate thechromatin environment and as such promotes OR expression when randomlyinserted in the genome.

Singular Gene Expression in the 4.M71Cα Line

In the 4.M71Cα line, the increase of OSNs expressing the M71 OR resultsin the coalescence of labeled axons onto one lateral glomerulus and onemedial glomerulus per OB. Surprisingly, these glomeruli weresignificantly larger (300μm) than typical glomeruli, providing theunique opportunity to answer a long-standing question in the field ofolfaction: What is the effect of increasing axonal input to a singleglomerulus on odor responses and behavior? To contrast the effect ofincreasing choice of the M71 OR in the 4′M71Cα line on the expressionlevel of the “endogenous” M71 OR and on axon guidance and identity, thisline was crossed to M71-IRES-tauGFP mice, gene-targeted at the M71 locus(M71G, 129 strain). A new mouse line was established that is eitherhomozygous for M71G (M71G−/−, henceforth known as WT) or both homozygousfor M71G and hemizygous or homozygous for the M71− IREStauCherrytransgene (4.M71Cα−/0 or −/−; M71G−/− henceforth known as 4.M71Cα Hemior Homo). Using coronal cryosections of the MOE of these 4.M71Cα hemianimals a total of 4754 mCherry-positive cells were counted expressingthe M71 4×21 transgene and 221 GFP-positive cells; none of the red cellsco-express the green marker in the MOE, suggesting that this 4×21 M71transgene maintains monogenic expression. In addition, confocal imagingof the OB of these animals reveals co-convergence of mCherry-positiveand endogenous GFP-positive axons onto the same isotypic M71 glomerulus,showing that OSNs expressing the M71 transgene have the same axonalidentity as endogenous M71 OSNs. Axonal identity strictly correlateswith the most abundant OR expressed in an OSN, thus it is unlikely thatother ORs are enriched in 4.M71Cα OSNs. Moreover, it is unlikely thatthe level of M71 RNA transcripts is elevated in the 4.M71Cα OSN as theycoalesce with M71G axons; this is consistent with previous observationsthat axons of OSNs expressing MOR23 from the 9×19 transgene co-convergeinto the same glomeruli as axons of OSNs expressing MOR23 from theendogenous locus and the knowledge that subtle changes to OR levels havedramatic affects on axon identity.

In Vivo Synaptophluorin (SpH) Imaging of Glomerular Activity

Because the M71 OR is dorsally expressed in the olfactory bulb, anycloned OR using the M71 transgenic backbone will coalesce its axons ontodorsally located glomeruli, which makes them accessible for opticalimaging. Optical imaging was therefore used to functionally characterizetransgenic M71 projections in the OB. To examine their functionalproperties odor-evoked activity was imaged from the OBs of miceexpressing the genetically encoded activity reporter SpH in all matureOSNs. Simultaneously recordings were obtained from mice that are eitherhemizygous or homozygous for 4.M71Cα (gene-targeted M71-GFP is outcrossed) and heterozygous for SpH and identify red M71 glomeruli. Sevendifferent M71-selective ligands were used from ex vivo analysis ofgenetically defined M71 OSNs in gene-targeted mice: three of theseligands produced responses by SpH imaging: Acetophenone (ACP), 4-methylacetophenone (4MACP) and 2,4-dimethyl acetophenone (24dMACP) whendelivered at different odor dilutions varying between 0% and 15%. Themaximum change in fluorescence [(dF/F)_(max)] per odor concentration wascalculated for each compound over a 14 s trial period having a pre-odor(3 s), odor (4 s) and post-odor (4 s) acquisition. dF/F values for allglomeruli were calculated with a standardized 50 μm window, which isfully contained in all imaged glomeruli of both hemi and homo 4.M71Cαanimals. (FIG. 2A, FIG. 2B and FIG. 2C: Each data point represents theaverage (dF/F)_(max) of 4-16 glomeruli imaged). Triangles indicatedresponses from hemizygous animals, circles indicate homozygous animalsand asterisks indicate homozygous responses that are significantlyhigher than hemizygous response when the animals are presented with thesame odor concentration. All imaged mice are heterozygous for omp-SpH.

M71 ligands show the same efficacy but different apparent affinity inhemizygous 4.M71Cα animals. In hemizygous animals, the average highest(dF/F)_(max) is 3.49% for ACP is (n=6 at 1.77 uM), 4.48% for 4MACP (n=9at 0.69 uM) and 4.15% for 24dMACP (n=7 at 0.66 uM). These results showthat all three compounds cause a similar efficacy [maximum responseobtained by a compound, i.e. average (dF/F)_(max)] in terms of M71 ORactivation (A one-way ANOVA comparing the highest (dF/F)_(max) valuesbetween Hemi animals did not reveal any significant differences).However, the apparent affinity of the M71 OR for 4MACP and 24dMACP issignificantly higher than ACP since the latter two compounds startactivating the M71 glomerulus at a lower concentration (FIG. 2). Forexample, when delivered at the same concentration (i.e. ˜0.70 uM),efficacy for ACP is 0.73% (n=4). This is significantly lower than theefficacy for both 4MACP (4.48%, n=9, Student's t-test, p<0.0001) and24dMACP (4.15%, n=7, Student's t-test, p<0.0001). Hence, similar odorefficacy is observed at lower concentration for both 4MACP and 24dMACPwhen compared to ACP responses in the Hemi animals.

Doubling the number of OSNs expressing M71 changes the efficacy for bothhigh- and low-affinity ligands. In homozygous animals, the highestefficacy for ACP is 4.57% (n=14 at 2.66 uM), 3.82% for 4MACP (n=16 at0.69 uM) and 4.92% for 24dMACP (n=7 at 0.46 uM). When comparinghemizygous and homozygous animals, the dose-responses do not differsignificantly for 4MACP. For 24dMACP, however, a significant increase inefficacy was observed at 0.44 uM (2.92% (n=4) vs. 4.92% (n=7), Student'st-test, p=0.0075), suggesting that doubling the number of OSNs thatchoose to express M71 changes the efficacy of this ligand when presentedat this particular concentration. In addition, the average efficacy (ofall concentrations and odors) is significantly different between 24dMACPand 4MACP in homozygous animals (4.92% vs 3.83%, Student's t-test,p=0.0272), even though they show practically the same dose-response inthe hemizygous animals (FIGS. 2A-2C). Doubling the number of neuronsexpressing the M71 OR changes the efficacy of 24dMACP, thus empiricallyidentifying it as the higher-affinity ligand.

Interestingly, in the homozygous animals, when ACP is delivered at 0.71uM, the efficacy is 3.00% (n=6) which is significantly higher (Student'st-test, p=0.0189) than the response at the same concentration in thehemizygous animals (0.73%, n=4)), reflecting a change in apparentaffinity for this ligand. Moreover, at the highest ACP concentrationdelivered (i.e. 2.66 uM), the homozygous response is also significantlyhigher than the heterozygous response (4.57%, n=14 vs. 3.46%, n=9).

Given a defined number of OSNs expressing M71, saturation of theglomerular response is reached at certain concentrations of a givenodor. However, this saturation response is not determined by a specificsubset of OSNs expressing one OR type. The data show that doubling thenumber of neurons expressing M71 can increase the glomerular efficacy atspecific concentrations.

Ex Vivo Vs. In Vivo Ligand Profiling

In the proof of concept experiments, odors are delivered from theheadspace of odor saturator vials containing 99% pure odorants andflow-diluted with air (N₂) prior to delivery to the anesthetized animal.The molar vapor concentration (uM) reaching the animal's nose istherefore determined by odorant saturated vapor pressure (P_(s)), whichcan differ dramatically between odorants. For example, 15% air dilutionof ACP (P_(s)=0.3260 mmHg at 25° C.) generates 2.66 uM airconcentration, while 4MACP and 24dMACP (P_(s)=0.0849 and 0.0811,respectively) are delivered at 0.69 uM and 0.66 uM, respectively. Invivo responses were compared with previous in ex vivo data by analyzingseven different odors that did (benzaldehyde, ethylmalthol, 2 aminoacetophenone, ACP, 4-metoxyacetophenone, 4MACP and 24dMACP) or did not(methylbenzoate and menthone) elicit a M71 response through ex vivopatch clamping of dendritic knobs when delivered in liquid phase.Glomerular activity was only observed with ACP, 4MACP and 24dMACP. Thereare two reasons for these findings: (1) the odor is not delivered at ahigh enough concentration and/or (2) the odor may not generate a highenough activation within OSNs. The minimal P_(s) necessary to be able toelicit a response in vivo is found to be 0.0811 mmHg (which is the P_(s)of 24dMACP). 4-metoxyacetophenone (a high responder ex vivo), does notshow glomerular activation in our hands and has P_(s)=0.0133 (<0.0811)mmHg, nor does benzaldehyde (a low responder ex vivo), which has thehighest P_(s) of all odors tested (1.0100 mmHg at 25° C.). Hence it issuggested that, when the minimal P_(s) requirement is met, a minimumcurrent amplitude (pA) M71 response (ex vivo) is used for an odor toactivate glomeruli in the SpH imaging setup.

Odor Detection Threshold in a Two-Bottle Discrimination Behavioral Task

Sensitivity studies performed in Wistar rat neonates and adults haveshown that the highest sensitivity to an odor (lowest threshold)correlates with the highest OSN density in the MOE. Even though thetotal number of mature OSNs in the 4.M71Cα animals were not changed[qPCR shows that olfactory marker protein (omp) RNA levels are notchanged between 4.M71Cα and WT animals], the neuronal representation ofOSNs expressing M71 is increased. Therefore, it was desirable to assesswhether detection of the most robust M71 ligand is amplified in this M71line. An avoidance task was used in which the odorant 24dMACP is addedto drinking water and becomes an aversive stimulus associated withinjection of Lithium Chloride (LiCl), a compound known to induce gastricmalaise. After conditioning homozygous 4.M71Cα animals and their WTlittermates to water containing a 10-4 dilution of 24dMACP, the animalsare given the choice between plain water and water with decreasingconcentrations of 24dMACP (10⁻⁶, 10⁻⁶, 5×10⁻⁷ and 10⁻⁷). Note that alltested animals are homozygous for M71G, so even WT have a functional M71OR.

The results are presented as a preference index (PI) for the odorizedwater for each group of animals which is calculated as the amount of theodorized water consumed divided by the total amount of liquid (odor andno-odor water) consumed over a 24 hr time period (FIG. 3A). A PI of 50%reflects no preference of the animal. A 10⁻⁴ dilution of 24dMACPcorresponds to 646 μM, a concentration that can be easily detected byboth homozygous and WT animals and does not cause an aversive responseby itself (the PIs for drinking water containing 10⁻⁶ of 24dMACP fornaïve, unconditioned mice are as follows: WT: 60%+/−7.72 (n=5) and Homo:59%.+/−1.94 (n=5)). When presented with 10⁻⁴ and 10⁻⁶ dilutions, bothconditioned 4.M71Cα homo and WT animals show a clear aversion towards24dMACP reflected through an average PI for 24dMACP of about 10%.However, when 24dMACP is delivered at 10⁻⁷ all animals fail to detectthe presence of the odor with an average PI of 53.41% for the WT (n=4)and 62.67% for the homozygous animals (n=8). This suggested that if thesensitivity of these M71 MouSensors were changed, the threshold would bebetween 10⁻⁶ and 10⁻⁷. Indeed, the PI of the WT group is significantlyhigher than the PI of the mutants at dilution 5×10⁻⁷ (62.79% vs 26.12%,Student's t-test, p<0.05), suggesting that while the WT fail to detectthe odor at 5×10⁻⁷, the mutants still show aversion behavior and thusstill smell 24dMACP. Importantly, even the WT animals show a lower PIfor the odorized water at 10⁻⁴ than at 5×10⁻⁷(11.84% vs 62.79%,Student's t-test, p<0.05), indicating that the behavioral difference ismost likely a result of the increased ability of the mutants to detectthe odor rather than a learning deficiency in the WT.

To compare specific odorant sensitivity between the 5.M71Cα and the4.M71Cα line, the 2-bottle discrimination behavioral task was performedwith 24dMACP as described above (FIG. 3B). In summary, at dilution5×10⁻⁷ the average PI of the 5.M71Cα group (n=9) is 19.62%, which issignificantly (p<0.01) lower than the PI of the WT group (n=8; 55.32%).Even though the same but slightly more significant 0.3 log decrease inodorant detection threshold is observed in the 5.M71Cα line, it isimportant to mention that 5.M71Cα hemi animals were used which haveabout 1.6% of their OSNs expressing M71 (similar to the 4.M71Cα homoanimals with about 2% M71 expressing OSNs).

It is important to note that precise odor-thresholding based on numberof OSNs is not being performed.

M71 Mousensors do not have a Monoclonal Nose

The percentage of OSNs expressing M71 changed in our 4.M71Cα line (whichis also homozygous for M71G) was assessed. Using qPCR, the level ofM71-mCherry RNA was found to be significantly increased by about 12 fold(Student's t-test, p<0.01) when compared to M71-GFP within thehemizygous 4.M71Cα line and by about 45 fold (Student's t-test, p<0.01)when compared to M71-GFP in the WT (M71G−/−) line. This apparentdiscrepancy is explained by the fact that the expression of thegene-targeted M71-GFP is 3.89 fold higher in the 4.M71Cα Hemi animalswhen compared to the WT animals (Student's t-test, p<0.01). Thesenumbers suggest that increasing M71-mCherry RNA levels help stabilizethe endogenous M71-GFP RNA levels early in development in the 4.M71Cαline, instead of cannibalizing them, leading to an increase inendogenous M71 transcripts in the 4.M71Cα animals. This model issupported by the fact that olfactory neurons are interdependent inmaintaining axonal projections. In addition, the postsynaptic bulbarcircuitry necessary to convey olfactory sensory information to thehigher cortical regions remains intact, as glomerulus-like structuresmay form in absence of postsynaptic olfactory neurons. An existing YFP-gtransgenic mouse line (in which 30% of the postsynaptic mitral cells arelabeled in yellow) was crossed to the 4.M71Cα line to visualize mitralcell dendrites. Mitral cell innervation of the M71 glomerulus is normal,as dendrites from postsynaptic mitral/tufted cells are present.

The M71G allele is expressed in about 0.03-0.06% of the OSNs (about 3KOSNs in 5-10M total, i.e. lower than the average 0.1%. Therefore, theabove observations suggest that the 4.M71Cα Homo animals (used in thebehavior) have about 1-2% of OSNs expressing the M71 OR. In this regard,the animals are fundamentally different from other animals generated byaltering the neural representation of odors by decreasing expression ofmost ORs by 95% and replacing them with M71, creating animals with amonoclonal nose. Despite previous observation that most OSNs andglomeruli can be (weakly) activated by ACP, odor discrimination andperformance in associative learning tasks is impaired in these animals.One possible explanation for this behavior is that broad uniformactivation (such as in the monoclonal nose) may cause lateral inhibitionthrough intricate feedback mechanisms (at the level of detection and/orperception) and ACP may be detected as olfactory “noise”. In thisregard, many small glomeruli could be disadvantageous compared to fewervery large glomeruli (which are observed in our 4.M71Cα line) andpatterned activation may be necessary for signal detection. On the otherhand, by reducing the representation of endogenous OR genes by 20-fold,they may have ablated the high-affinity OR for ACP as well. (e.g.Olfr145, von der Weid et al., Nature Neuroscience, Large-scaletranscriptional profiling of chemosensory neurons identifiesreceptor-ligand pairs in vivo, 2015)

A Versatile Platform

The phrase “odorant receptor coding sequence” refers to G-ProteinCoupled Receptor (GPCR) that exhibits the following functions (1)odorant identification and olfactory signal transduction (2) maintenanceof first choice to block the expression of a second OR (3) promotion ofmaturation of the neuron (4) providing the axons with the ability togrow and (5) encryption of axonal identity to growing axons. Numerousexamples of odorant receptor transgenes are known to those skilled inthe art with M71 and OR1A1 merely being two examples. The disclosedmethods are applicable to a wide range of odorant receptor transgenes.

To assess the effect of further increasing the number of multimers as aubiquitous enhancer on the number of OSNs choosing to express any clonedOR, lines containing 5×21 enhancers for two mouse ORs, M71 and MOR122-2were created. All founders for 5.M71C (6/6) and one germline animal5.M71Cα show high levels of M71 expression (Table 1) and form glomeruliin the bulb. In addition, all five germline animals for 5.MOR122-2 showhigh levels of expression for MOR122-2. Moreover, when comparing 5.M71Cαwith 4.M71Cα, the data show adding an extra 21mer increases the numberof OSNs expressing the M71. This increased number appears to be a resultof multimerization and not of increased copy number of the transgene inthe genome (about 16 times for 5×21 M71 vs. about 15 for the 4×21 M71transgene. In addition, using coronal cryosections of the MOE of a 6.5weeks old 5.M71C Hemi animal a total of 52 green cells were countedamongst a large population of red cells and none of them coexpressed.These data show that adding five 21mers maintains singular geneexpression in OSNs. Based on current models of singular gene choice,this choice enhancer can still be suppressed in a large number ofneurons and should be a target of silencing in non-cherry cells. Thisdisclosure provides a model system for testing the role coding sequencesor other genomic sequences for its capacity to be silenced.

To further evaluate the versatility of this platform, the probability ofchoice of expression of a human OR was increased by cloning OR1A1 intothe 5×21 M71 transgenic backbone. Both germline animals (5.OR1A1C β andδ) show robust expression of the human OR1A1 receptor in the MOE andstable glomerular formation in the OB. qPCR reveals even a ˜12 foldincrease of OR1A1 RNA in the 5.OR1A1Cβ hemi animals when compared to theM71 RNA levels in the 4.M71Cα hemi line. Again omp levels were notchanged. Clearly, the representation of some ORs must be decreased toexplain this equilibrium. In the 4.M71Cα homo animals, a mere 2-logincrease in M71 RNA levels leads to a detectable 0.3-log change inthreshold. Therefore, without wishing to be bound to any particulartheory, it is believed that further increases in the neuronalrepresentation of specific ORs may decrease detection thresholds evenmore. However, a delicate balance must exist between cell number andsensitivity. This hypothesis was tested using the established two-bottlebehavioral avoidance task with the 5.OR1A1Cβ mice (FIG. 3B).Conditioning of 5.OR1A1Cβ hemi animals (n=12) was performed and their WTlittermates (n=7) to water containing a 10-4 dilution of (−)-Carvone, aknown OR1A1 ligand. For four consecutive days, animals were given thechoice between plain water and water with decreasing (10-4, 10-6, 10-7and 10-8) dilutions of (−) Carvone (FIG. 3C). Both groups (WT and Hemi)show clear aversion towards a 10⁴ dilution of (−)-Carvone (AVG PIWT=6.17% and AVG PI hemi=10.26%), reflecting successful conditioning.Both WT (AVG PI=55.52%) and Hemi (AVG PI=47.88%) cannot detect a 10⁻⁸dilution of (−)-Carvone. At 10⁻⁶ and 10⁻⁷ however, there is a cleardifference in detection between WT and Hemi (10⁻⁶: AVG PI WT=27.65% vs.AVG PI Hemi=11.41, Student's T-test p<0.01 and 10⁻⁷: AVG PI WT=53.88%vs. AVG PI Hemi=30.24, Student's T-test p<0.05). Importantly, even theWT animals show significantly lower PIs for the odorized water at 10⁻⁴than at 10⁻⁶ (6.17% vs. 27.65%, Student's t-test, p<0.01) and at 10⁻⁷(6.17% vs. 53.88%, Student's t-test, p<0.0001), again indicating thatthe behavioral difference is most likely a result of the increasedability of the mutants to detect the odor rather than a learningdeficiency in the WT (FIG. 3B). These findings indicate that expressingOR1A1 in about 40% of the OSNs translates into a 2-log difference in(−)-Carvone detection thresholds.

CONCLUSIONS

The disclosed transgenic platform is an invaluable tool to further studythe mechanisms of singular gene choice and odor coding in the olfactorysystem. Mechanistically, HD binding motifs could modulate theprobability of OR gene choice by attracting polymerase assembly factors,enhancing transcription initiation, or stabilizing transcriptionelongation. Placing HD elements in close proximity of each other willmost likely favor strong cooperative binding of transcription factors.In this regard, nine 21mers have been multimerized resulting in evengreater representation of ORs in the olfactory epithelium. This platformfurther enables one to dissect the mechanism of OR gene choice ornegative feedback on first choice using mutant OR sequences. Inaddition, the possibility to express human ORs in large numbers of mouseOSNs using the disclosed technology provides a breakthrough in vivoapproach to finally crack the olfactory code.

An example of a 5×21 annealed ultramer insertion into the NheI site(gctagc) is provided below. A forward ultramer comprising a five-repeatis given by ctagcACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACA TAACTTTTTAATGAGTCT(SEQ ID NO: 11). A reverse ultramer comprising a five repeat is given byctagAGACTCATTAAAAAGTTATGTeAGACTCATTAAAAAGTTATGTAGACTCATTAAAAAGTTATGTAGACTCATTAAAAAGTTATGTg (SEQ ID NO: 12).

A M71 promoter sequence (485 bp plus gctagc) is given by:

(SEQ ID NO: 13) gctagcTCATGATGCATCATGGTGACTAGCTAACAATATGTAATAACCATCATGGCACTGAGAATGATGTTGCTGGTAGTTACTGTGGTGCCTATTGAGATGAAAATGACAATAATGATAATACATCAGTAGTAAAGGTGATAGCCATGGTCATAATGGTAATGATGATAAGGATGGGTGGGTAGTGGTGATGGATGGTTGGTGGTGGTGGTGGTGATGGTAGTGGTAGGGGGGTAATGGTGGTGGTAACGGCTCTGTTGATGCTAAATTGTTCATTGCCCCATTATATTCTAAGTTTCTGAAACTGAAAGATGACTTTTACAGATAAAAGAAGAATTAACACACTTGGGAAATAAAACATGATTCACAGAACAAGAGAAAACATGAACTAATTGTTACTTTAGAGACAACAATGGTCTCTAGAGTGACTATATCCCAGGAGATGATCCACACACACACACACATATATATAAACAGAACCCCCAATTTTT.

The 5×21 M71 transgene promotor is given by

(SEQ ID NO: 14) gctagcACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTACATAACTTTTTAATGAGTCTctagcTCATGATGCATCATGGTGACTAGCTAACAATATGTAATAACCATCATGGCACTGAGAATGATGTTGCTGGTAGTTACTGTGGTGCCTATTGAGATGAAAATGACAATAATGATAATACATCAGTAGTAAAGGTGATAGCCATGGTCATAATGGTAATGATGATAAGGATGGGTGGGTAGTGGTGATGGATGGTTGGTGGTGGTGGTGGTGATGGTAGTGGTAGGGGGGTAATGGTGGTGGTAACGGCTCTGTTGATGCTAAATTGTTCATTGCCCCATTATATTCTAAGTTTCTGAAACTGAAAGATGACTTTTACAGATAAAAGAAGAATTAACACACTTGGGAAATAAAACATGATTCACAGAACAAGAGAAAACATGAACTAATTGTTACTTTAGAGACAACAATGGTCTCTAGAGTGACTATATCCCAGGAGATGATCCACACACACACACACATATATATAAACAGAACCCCCAATTTT T.

Method of High-Throughput Screening for an Odorant Ligand

Also disclosed in this specification to a method to measure biochemicalchanges in this OR signal transduction cascade within olfactory neuronswhen challenged with odors.

When odors bind odorant receptors (ORs), a signal transduction cascadeensues whereby the Golf alpha subunit becomes activated through bindingto GTP. Subsequently, Golf-GTP activates adenylate cyclase III, whichcatalyzes a conversion of ATP into cAMP. The reaction terminates withthe cleavage of GTP to GDP creating Golf-GDP.

Odorant receptors are notoriously hard to express in vitro. Becauseheterologous expression systems are lacking, most ORs remain orphans,meaning that their correspondent ligands are unknown. Thus, theolfactory code has to be studied in vivo, however this process ishampered because each OR gene is expressed in only 0.1% of the OSNs,making it difficult to correlate specific ligands to their cognate ORs.

One group claims to have found a way to express ORs in vitro using achaperone molecule. Duke University licenses the use of this patentedtechnology exclusively to ChemCom, a small Belgian company. (Saito etal., Cell, Vol. 119, 679-691, Nov. 24, 2004) Therefore, all big playersin the fragrance industry have to find a “work around” to express ORs.Currently, research perfumers and human psychophysics empiricallyvalidate the odor quality of newly synthesized molecules. This expensivemethod is ineffective because less than 1% of the manufactured newmolecules find their way to the market per year, largely due to the lackof a high-throughput screening platform that can reduce the number ofcandidate molecules to be tested by humans and ultimately reduce thepipeline cost.

The methods described elsewhere in this specification provide a platformthat can express any odorant receptor, including human ORs, in largequantities in the mouse brain. The main olfactory epithelium thatcontains these transgenic OR expressing olfactory sensory neurons can beeasily dissected and lysed.

ORs are G-protein coupled receptors (GPCR) that, when activated by theirligand, initiate a signaling pathway that involves the conversion GTP toGDP and ATP into cAMP. Therefore, an increase in GDP/cAMP or a decreasein ATP is an indicator of ligand binding activity.

Because the biosensors described in this disclosure express the OR ofinterest in the majority of their cells any measurable increase inGDP/cAMP (or decrease in ATP) by a specific ligand reflects activationof the transgenic OR. The method described herein provides aquantitative measurement of increased GDP/cAMP decreased ATP afterspecific odorant activation as a means for high-throughput screening ofscent molecules (agonists and antagonists) using any genetic platformthat increases the neuronal representation of specific ORs.

Molecular kits that quantify these biochemical changes are commerciallyavailable. Levels of cAMP and ATP can be measured by existingtechnologies, such as luciferase kits or by direct coupling of ATP toQuenchers and/or fluorophores. In one embodiment, a microfluidic chip isused to measure activity.

Applications

One benefit of a genetic approach to create biosensors is that theincreased sense of smell will be genetically defined and thusinheritable. For example, offspring of a TNT mouse will also carry theenhanced sensory ability. In addition, transgenic mice have fastbreeding times and maturation rates; the gestation period for a mouseranges from 18-22 days. Additionally, animals with an inherent enhancedsense for specific smells are anticipated to have dramatically decreasedtraining time and enhanced the efficiency.

The disclosed methods are useful in multiple applications includingdisease diagnostics, drug prevention, search and rescue and pathogendetection.

Examples of biosensors useful in disease diagnostics include biosensorsthat identify tuberculosis (TB) in human saliva samples. Globally, theinfectious disease TB is a leading cause of death, second only to AIDS.M. tuberculosis, the bacteria that causes TB, produces two specificodors (Methyl nicotinate and Methyl 4-anisate) which are unique to thebacteria. ORs sensitive to these odors can be identified and used in ourtransgenic platform to create TB biosensors.

Examples of drug prevention biosensors for contraband searches includecocaine sensitive biosensors. Examples of search and residue biosensorsinclude biosensors that provide police assistance to help locate victimsafter natural disasters (e.g. cadaver dogs). Examples of biosensors forpathogen detection in livestock include biosensors that identify ofanimals infected with cattle virus.

Materials and Methods

Subjects: Mice used in this study were bred and maintained in theLaboratory Animal Facility of Hunter College, CUNY. The Hunter CollegeIACUC approved all procedures. Animal care and procedures were inaccordance with the Guide for the Care and Use of Laboratory Animals(NHHS Publication No. (NIH) 85-23).

Genotyping

Presence of 4×21-M71-IRES-tauCherry transgene was assessed by PCR byscoring for the mCherry gene with the following primers: FWD: 5′CCCTGGACAACATCACAC 3′ (SEQ ID NO: 3) and REV 5′CCCTCCATGTGCACCTTGAAGCGCA 3′ (SEQ ID NO: 4). To distinguish betweenhemizygous and homozygous 4.M71Cα animals we used a qPCR method.Presence of the M71-IRES-tauGFP gene targeted allele was determined byusing primers to detect GFP: FWD 5′ CCCTGGACAACATCACAC 3′ (SEQ ID NO: 3)and REV 5′ CGTTTACGTCGCCGTCCAGCTC 3′ (SEQ ID NO: 5) and primers todetect the WT M71 allele with FWD 5′ CCGCACTGGACAAAACACTGAGGAG 3′ (SEQID NO: 6) and REV 5 ‘CTGTTTCCTGTTCAGAGTTGGGTG 3’ (SEQ ID NO: 7),allowing us to distinguish between WT, Hemi and Homo animals.

Olfactometry: Odors (purchased form Sigma Aldrich) were delivered usinga custom built olfactometer controlled by an Arduino board with customshields operating Teflon solenoids (Neptune Research) and connected totwo Mass Flow Controllers (Alicat Scientific, Inc) to dilute the cleanair (N₂, max flow is 2 Lpm) and to dilute the odorized air (max flow is300 SCCM). Nitrogen (N₂) was used as the vapor carrier to avoidoxidation. Odor concentrations are expressed a % dilutions of saturatedvapor and as molar saturated vapor concentration (μM s.v.), calculatedusing published vapor pressures at 25° Celsius (US EPA, EstimationPrograms Interface Suite, v 4.0. Flow diluted odors were delivered at 2Lpm mixed prior to the delivery site with air odorized with varyingdilutions of odorant taken from the saturated headspace of pre-cleanedamber vials with white polypropylene closures and septum (J.G. Finneran)containing 99% pure odorants. To avoid contamination of theolfactometer, odorized air was never passed through the Teflon valves.Instead, odors were mixed with N₂ in T-shaped mixing chambers (NeptuneResearch) and delivered to the animal's nose using an arduino controlledvacuum system. Odorized streams carrying different odors did not comeinto contact. Output of the olfactometer was calibrated using the tracerodor Pinene and a photoionization detector (PID; Aurora Scientific).

In vivo SpH Imaging: Mice were anesthetized using Ketamine (100mg/kg)/Xylazin (5 mg/kg) and maintained with Isoflurane (2.5% in 02) andimmobilized using a stereotactic head holding device (Narishige, Inc.).Optical signals for SpH and mCherry were recorded using an ANDOR Neo 5.5sCMOS camera connected to a NIKON AZ100 epifluorescence microscope (NISElements) with a 4× objective (numerical aperture 0.4). Excitationwavelengths of 475 nm for SpH and 575 nm for mCherry and emission of 520nm for SpH and 635 nm for mCherry were used to obtain images of thedorsolateral bulb through thinned skull overlaying the bulb of a freelybreathing animal. Each imaging trial consisted of a pre-odor (3 s), odor(4 s) and post-odor (4 s) acquisition, with a total acquisition time pertrial of 14 s (including 3 s of valve switch delay). Using serial code,the arduino controlling the olfactometer was integrated in the NISElements software, so that the entire image acquisition sequence andflow-diluted odor delivery is controlled by the microscope software.

Image Processing: Calculation of image response statistics was performedusing custom code written in Matlab (Mathworks). Image frames wereexported from the microscope NIS Elements AR Software and imported intoMatlab. Frames were spatially bandpass filtered (Gaussian kernel;fspecial; high pass sigma=500 um; low pass sigma=10 um). Image frameswere divided by the mean of the frames collected prior to odor onset(resting F) to correct for light distribution across the image. Togenerate glomerular activity maps (dF/F), divided pre-odor frames weresubtracted from divided post odor frames. Regions of interest (ROIs;circles of radius 50 μm) were selected from manual examination of thepost odor frames. Activity time-courses within each ROI were generatedby averaging all pixels falling within the ROI for each frame. Theresulting traces were exported into Excel for further analysis.

Olfactory behavior test: A two-bottle discrimination test was performedas previously described. Since the mice are tested for the ability todetect a single odor in drinking water at increasingly lowerconcentrations, this discrimination assay is also considered a detectionthreshold test. Mice 8 weeks old were individually housed and given foodad libitum but restricted access to saccharin-phthalic acid solution(2.1×10⁻² M sodium saccharin and 10⁻³M phthalic acid pH 6.5) for 1 htwice a day for two days before the assay begins. This ensures micewould commence drinking the solution during the conditioning. On day 3(conditioning day) mice are exposed to sodium saccharine phthalic acid(SSPA) solution with 10⁴ dilution of odorized water for 10 minutes.Immediately after, they are injected with lithium chlorideintraperitoneally (15 μl/g body weight of a 0.6 M solution) to inducethe aversive malaise and lethargy state. After two hours mice arereturned to their home cage and given access to two bottles of drinkingwater; they are given the choice between SSPA solution containing a 10⁴dilution of the odor versus the non-odorized SSPA solution. During thefollowing 3 days, every 24 hours, the location of the bottles isreversed and the concentration of the odorized solution is decreased to10⁻⁶, 10⁻⁷ and 0.5×10⁻⁶, respectively. Every day both bottles areweighed to determine the amount of liquid consumed. A preference indexwas calculated as the amount of odorized solution consumed divided bythe total amount of water solution consumed for each mouse for every 24hour test period at each odor concentration. Student's t test wasperformed to test statistical significance, assuming two-taileddistribution and two-sample unequal variance. Values are mean±SEM andare plotted on a LOG scale. Animals (both WT and Homo) that did not seemto be conditioned after LiCl injections (meaning showing a PI for24dMACP higher than 20% at 10⁻⁴) were excluded from our analysis (i.e. 4WT and 2 homo).

RNA extraction and cDNA synthesis: 4.M71Cα Hemi animals were sacrificedand the olfactory epithelium tissue was dissected on a mixture of iceand dry ice. Tissue was snap frozen in liquid N₂ and stored at −80° C.RNA was isolated using the RNEASY® Mini columns (Qiagen, Cat #74104)with DNase digestion according the manufacturer's protocol. Tissue washomogenized with mortar and pestle in lysis buffer provided by the kitcontaining 10 μL ß-mercaptoethanol. The concentrations of the isolatedRNA samples were measured using a NANODROP® ND-1000 Spectrophotometer.Only RNA samples with a 260/280 ratio between 1.8 and 2.1 wereconsidered for further processing. After RNA extraction, an additionalDNase digestion was performed with a TURBO DNA-FREE™ DNase Treatment andRemoval (Life Technologies, cat # AM1907) kit to remove all genomic DNA(gDNA) from the sample. First-strand cDNA was synthesized usingSUPERSCRIPT″ III First-Strand Synthesis System for RT-PCR (LifeTechnologies, cat #18080-051). First-strand cDNA was diluted 1:5 to atotal volume of 100 μl. Genomic contamination of the generated cDNA waschecked with regular PCR using with 2 primers located in exonsoverspanning a small intron of the HipkII gene.: FWD 5′TGTGAGGCAATTGACATGTGG 3′ (SEQ ID NO: 8) and REV 5′ TACGGTGAGTCTGTGTCAC3′ (SEQ ID NO: 9).

Quantitative Real-Time PCR: qPCR was performed according to the latestMinimum Information for Publication of Quantitative Real-Time PCRExperiments (MIQE) guidelines (8) using hydrolysis probe sets. RNA wasisolated form olfactory epithelial tissue from 6.5 week old littermates(4.M71Cα Hemi and WT, n=3). Amplification efficiencies of theprimer/hydrolysis probe sets were calculated based upon the generationof standard curves using a 2-fold cDNA (for Gapdh, Pgk1, Tfrc, Rn18S,Omp) or plasmid DNA (for mCherry, GFP) dilution series. PCR efficiencieswere calculated using the slopes of the standard curves according to thefollowing formula: PCR E=10^(−1/slope)−1. The linear dynamic range was 4dilutions and Taqman IDs, standard curve slopes, PCR efficiencies, r²values and linear dynamic range for all amplicons analyzed is summarizedin Cell Reports 16, 1115-1125 (2016). The qPCR reaction mixture (10 ul)contained 5 ul TAQMAN® Gene Expression Master Mix (Applied Biosystems),0.5 ul of the TAQMAN® probe (Applied Biosystems) and primer mix, 2.5 ulof deionized and 2 ul of cDNA. qPCR reactions were set up manually usinga Matrix Electronic Multichannel Pipette with 12.5 ul Impact 384 Tips(Thermo Scientific, Hudson, N.H., USA, cat#7421) and the reactions werecarried out in white 384 well-plates (E&K Scientific, Inc., Santa Clara,Calif., USA, cat#486384) covered with ThermaSeal optical covers (ExcelScientific, Inc., Victorville, Calif., USA, cat# TSS-RTQ-100)). Reactionwere run on a Roche Lightcycler 480 using the following cyclingconditions: an initial denaturation step at 95° C. for 10 s, followed bya 45-cycle amplification step of 95° C. for 10 s, 60° C. for 30 s and72° C. for 1 min and a final cooling step at 40° C. for 10 s. The Cqvalue for the no template controls (NTC) was >30 for all hydrolysisprobe sets. qPCR levels for each hydrolysis probe set were normalized to4 different reference genes Gapdh, Pgk1, Tfrc and Rn18S (selected basedon (9)), that were stably expressed between WT and 4.M71Cα Hemi animalsas shown by their coefficient of variance (CV). The CV is defined as theratio of the standard deviation to the means and it is a usefulstatistic for comparing the degree of variation from one data series toanother, even if the means are drastically different from each other.Reference genes with a CV lower than 25% were considered stablyexpressed between WT and 4.M71Cα hemi. CV's were as follows: Gapdh:23.95%; Pgk1: 21.65%; Tfrc: 18.52% and Rn18S: 24.61%. We used thecomparative Ct method or 2^(−ddCt) method to calculate the normalizedrelative quantitative (NRQ) values for each sample per gene and the folddifference in RNA expression levels.

Mitral Cell labeling: Male YFP-g mice (Strain: 014130, JacksonLaboratory) were crossed with female hemizygous 4.M71Cα (also M71G−/−)mice to generate compound mutant mice. Male offspring that were positivefor both YFP and mCherry were euthanized at 8-12 weeks of age andolfactory bulb tissue was processed for histological imaging. Imageswere collected on a LSM510 confocal microscope (Carl Zeiss) usingobjectives, Fluar, 10×N.A. 0.5 and PlanNeofluar 40×, N.A.1.3.

Immunohistochemistry: Male hemizygous 4.M71Cα (also M71G−/−), 8-12 weeksof age, were perfused transcardially with 1×PBS followed by 4% PFA. Theolfactory bulbs were dissected, embedded in gelatin and postfixedovernight in 2% PFA+50% sucrose for cryoprotection. Olfactory bulbs werecryostat sectioned at 50 coronally, and processed for immunofluorescencestaining. Primary antibodies: TH (1:2500, Novus), 5T4 (1:250, R&DSystems), Calretinin (1:2500, Millipore), Calbindin (1:2000, Chemicon)and CCK (1:1000, Sigma) for 2 days at 4 degrees. All sections followedby secondary antibody containing Cy5 (1:600, Jackson ImmunoResearch)incubated at room temperature for 4 hours. Images were collected on aLSM510 confocal microscope (Carl Zeiss) using objectives, Fluar, 10×N.A.0.5 and PlanNeofluar 40×, N.A.1.3.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal language of the claims.

What is claimed is:
 1. A method of producing a genetically modifiednon-human vertebrate, the method comprising inserting DNA into a genomeof a non-human vertebrate using a vector, the vector comprising: atransgene backbone; at least three sequential repeats of a DNA sequencewith two complete ds B-DNA turns, the DNA sequence being at least 90%homologous with 5′ ACATAACTTTTTAATGAGTCT 3′ (SEQ ID NO: 1); atranscription start site (TSS) disposed downstream of the at least threesequential repeats; an odorant receptor coding sequence disposeddownstream of the transcription start site, the odorant receptor codingsequence coding for a predetermined odorant receptor; wherein thenon-human vertebrate, after the step of inserting, develops a mainolfactory epithelium (MOE) comprising multiple olfactory sensory neurons(OSNs), wherein between 10% and 95% of the OSNs express thepredetermined odorant receptor.
 2. The method as recited in claim 1,wherein the odorant receptor coding sequence is a M71 odorant receptorcoding sequence.
 3. The method as recited in claim 1, wherein thenon-human vertebrate is a mouse.
 4. The method as recited in claim 3,wherein the odorant receptor coding sequence is a M71 odorant receptorcoding sequence.
 5. The method as recited in claim 1, wherein thenon-human vertebrate is a rat.
 6. The method as recited in claim 1,wherein the non-human vertebrate is a dog.
 7. The method as recited inclaim 1, wherein the DNA sequence is at least 95% homologous with SEQ IDNO:
 1. 8. The method as recited in claim 1, wherein the DNA sequence isSEQ ID NO:
 1. 9. The method as recited in claim 1, wherein there are atleast four of the sequential repeats.
 10. The method as recited in claim1, wherein the transgene backbone is an M71 odorant receptor (OR)transgene backbone.
 11. A vector for genetically modifying a vertebrate,the vector comprising: a M71 odorant receptor (OR) transgene backbone;at least three sequential repeats of a DNA sequence that is at least 90%homologous with 5′ ACATAACTTTTTAATGAGTCT 3′ (SEQ ID NO: 1); atranscription start site (TSS) disposed downstream of the at least threesequential repeats; an odorant receptor coding sequence disposeddownstream of the transcription start site, the odorant receptor codingsequence coding for a predetermined odorant receptor; wherein thevertebrate develops an olfactory epithelium that expresses between 10%and 70% of the predetermined odorant receptor.
 12. The vector as recitedin claim 11, wherein there are at least four of the sequential repeats.13. A genetically modified non-human vertebrate comprising germ cellsand somatic cells that contain an insertion of DNA within a 10 kbproximity of a transcription start site (TSS) of an odorant receptortransgene, such that the insertion causes the vertebrate to overrepresent a predetermined odorant receptor corresponding to the odorantreceptor transgene relative to a corresponding vertebrate that lacks theinsertion, wherein the DNA comprises at least three sequential repeatsof a DNA sequence with two complete ds B-DNA turns whose primarystructure is at least 90% homologous with ACATAACTTTTTAATGAGTCT (SEQ IDNO: 1).
 14. The non-human vertebrate as recited in claim 13, wherein thenon-human vertebrate is a mouse.
 15. The non-human vertebrate as recitedin claim 13, wherein the non-human vertebrate is a rat.
 16. Thenon-human vertebrate as recited in claim 13, wherein the non-humanvertebrate is a dog.
 17. The non-human vertebrate as recited in claim13, wherein the predetermined odorant receptor is over represented by afactor of at least fourfold.
 18. The non-human vertebrate as recited inclaim 13, wherein the predetermined odorant receptor is over representedby a factor of at least tenfold.
 19. The non-human vertebrate as recitedin claim 13, wherein the DNA sequence is at least 95% homologous withSEQ ID NO:
 1. 20. The non-human vertebrate as recited in claim 13,wherein the DNA sequence is SEQ ID NO:
 1. 21. The non-human vertebrateas recited in claim 13, wherein there are ten or fewer of the sequentialrepeats.
 22. The non-human vertebrate as recited in claim 13, whereinbetween 10% and 70% of an olfactory epithelium of the vertebrateexpresses the predetermined odorant receptor.
 23. A method ofhigh-throughput screening for an odorant ligand, the method comprisingsteps of: exposing a candidate odorant ligand to an olfactory neuron orcilia of a biosensor, the biosensor comprising a plurality of cells withneurons in an olfactory epithelium wherein greater than 1% of theneurons in the olfactory epithelium are expressing an odorant receptor(OR) through a singular gene choice enhancer nucleotide sequence with atleast three repeats of a DNA sequence that is at least 90% homologouswith 5′ ACATAACTTTTTAATGAGTCT 3′, the at least three repeats beingdisposed within 10 kb from a predetermined odorant receptor; monitoringthe olfactory neuron or the cilia for a change in expression of GDP,cAMP and/or ATP, wherein an increase corresponds to successful bindingof the candidate odorant ligand to the predetermined odorant receptor.